<p>Immediate hypersensitivity (allergy) is a very common disorder, which may<br />develop after exposure of an individual to an antigen. Intestinal hypersensitivity<br />to luminal antigens has been postulated as a possible cause or triggering<br />mechanism in the pathogenesis of ulcerative colitis, Crohn's disease, eosinophilic<br />gastroenteritis, celiac disease and peptic ulcer. The mechanism by which<br />individuals became sensitized is not known but naturally occurring adjuvants<br />(bacteria and their products) may be crucial for the development of<br />hypersensitivity. In my studies, I investigated in the role of pertussis toxin, a<br />product of Bordetella pertussis, in intestinal hypersensitivity, since pertussis<br />vaccine containing attenuated bacteria was used previously for the induction of<br />anaphylaxis in experimental animals. Pertussis toxin has the enzymatic activity<br />of ADP-ribosyltransferase. which can block the function of some G proteins,<br />important elements in the transduction of signals into the cell. I sensitized rats<br />or mast cell-deficient mice with ovalbumin (OVA) plus recombinant wild type<br />pertussis toxin (wPT) as adjuvant. The reaction to secondary antigen challenge<br />was evaluated 14 days later by determining changes in short-circuit current (Isc, indication of net ion transport) in small intestinal segments mounted in Ussing chambers. Other parameters measured included evaluation of antibody levels in the circulation and histological enumeration of mast cells in the intestinal mucosa.</p> <p>Sensitization of rats with OVA and wPT resulted in enhancement of the<br />jejunal ion secretory response to secondary antigen challenge as compared to<br />rats sensitized with OVA. Injection of doses of wPT as low as 10 ng with OVA<br />caused significantly elevated secondary responses to OVA, while 50 ng wPT<br />resulted in the maximal response. The responses to OVA challenge were<br />enhanced 18.7 fold when OVA was added to the luminal side of the tissues<br />while on serosal side this effect was enhanced 2.2 fold in sensitized rats. The<br />induction of hypersensitivity by wPT adjuvant was dependent on the enzymatic<br />activity of wPT. since the enzymatically inactive mutant (mPT) did not influence<br />responses to secondary antigen challenge. This suggests that ADP-ribosylation<br />of G proteins is involved in the elevation of hypersensitivity by wPT.</p> <p>Two classes of antigen-specific antibodies, IgE and IgG₂s., were measured<br />in the circulation of sensitized rats. The levels of both antibody isotypes were<br />increased at day 14 in wPT but not mPT plus OVA injected rats, which indicates<br />that enzymatically active wPT influences antibody production. The role of<br />particular classes of antibodies was examined by passive sensitization of naive<br />animals with serum from actively sensitized rats. Ion transport responses to<br />secondary antigen challenge of intestine were present in those animals.<br />Blockage of IgE receptors (with myeloma IgEI prior to passive sensitization<br />showed that the response to OVA was totally abolished. This finding shows that<br />in this model of hypersensitivity. IgE antibodies are crucial for the response to antigen. Experiments on mast cell-deficient mutant mice revealed that mast cells<br />are a necessary element for the responses to antigen. Additionally, the number<br />of stainable mast cells in rat intestinal mucosa was significantiy increased by<br />40% on day 14 in wPT but not mPT plus OVA injected rats. This indicates that<br />wPT-induced increases in mast cell numbers are partially responsible for elevated<br />hypersensitivity responses to antigen. Experiments with the neural toxin,<br />tetrodotoxin (TTX), showed that responses to luminal (but not serosal) OVA<br />were neurally regulated in that these responses to OVA were diminished by 66%<br />after treatment of the tissues with TTX.</p> <p>Evaluation of tne responses to OVA at different days after primary<br />sensitization with OVA plus wPT revealed that the responsiveness to antigen<br />appeared between day 3 and 7 after sensitization, and it was still present on day<br />228. In rats sensitized with OVA, the responsiveness to OVA was highest on<br />day 7 and than it decreased very rapidly, showing no responses on day 56.<br />Evaluation of IgE levels in rats demonstrated a similar pattern: the IgE levels were<br />detectable for a long time in wPT plus OVA treated rats but only on day 7 in<br />OVA treated rats. This suggest that elevated IgE levels caused by wPT may<br />account for the long lasting hypersensitivity responses.</p> <p>My data showed that wPT is a very potent adjuvant for hypersensitivity<br />reactions and that the mechanism involves elevation of antigen-specific<br />antibodies, increases in the number of mucosal mast cells and enhanced neurally-mediated<br />antigen uptake.</p> / Doctor of Philosophy (PhD)

<p>Ryanodine, a neutral alkaloid, is a widely used pharmacological tool in the studies<br />of muscle excitation-contraction coupling. The specific binding sites for ryanodine have<br />been identified to exist on sarcoplasmic reticulum (SR) in both skeletal muscle and<br />cardiac muscle. The ryanodine receptor has been purified from different tissue types. The purified ryanodine receptor from skeletal muscle was found to be identical with foot<br />structure, a protein spanning the gap between t-tubule and SR membrane. The ryanodine<br />receptor was also found to have Ca²⁺ channel activity, a Ca²⁺-induced Ca²⁺ release channel.<br />In functional studies with isolated SR vesicle or single channel, ryanodine was found to<br />have dual effects on Ca²⁺ channel. At lower concentrations, ryanodine locked Ca²⁺ channel<br />in the open state, but fully closed the channel at higher concentrations. In smooth muscle,<br />ryanodine was also found to affect intracellular Ca²⁺ movement in the functional studies<br />using intact tissue. However, direct evidence was not available for the presence of<br />ryanodine receptor in smooth muscle before this research was carried out.</p> <p>In the present study, a high affinity binding site was found located on SR<br />membranes of rat vas deferens (RVD) smooth muscle (Kd=5.6 nM, Bmax=435 fmol/mg).<br />The ryanodine receptor in smooth muscle shared many similarities with that of skeletal<br />muscle, but was not identical. The time required for [³H]ryanodine binding to microsomal<br />fraction was about two hours to reach a steady state, and [³HJryanodine could be dissociated from its binding site by 20 fold dilution. However, the dissociation was very<br />slow and incomplete when initiated by excess ryanodine. The [³H]ryanodine binding in<br />smooth muscle was Ca²⁺ dependant. The affinity was increased with increased Ca²⁺<br />concentrations, but the Bmax was unchanged. The [³H]ryanodine binding also increased<br />with higher ionic strength and higher osmolarity, but later has less effect. Many factors<br />that affect Ca²⁺-induced Ca²⁺ release (CICR) channel activity were also found to affect [³H]ryanodine binding in my study; e.g., both Mg²⁺ and ruthenium red inhibited binding<br />and caffeine potentiated it, especially in the presence of a low Ca²⁺ concentration. In the<br />present study, I also showed that varied levels of [³H]ryanodine binding site existed among different smooth muscles. This variation was not correlated with the density of innervation or the SR content of different smooth muscles.</p> <p>In dog mesentery artery smooth muscle, a low affinity binding site (Kd=269 nM) was also identified, in addition to the high affinity binding site.</p> <p>In the present study, I also tried to show functional effect of ryanodine on the<br />CICR channel using subcellular membrane vesicles from vas deferens. Ryanodine, at<br />higher concentrations, inhibited oxalate-stimulated Ca²⁺ uptake, an effect which was observed as early as 5 minutes after uptake was initiated. This inhibitory effect was partially additive to that of cyclopiazonic acid (CPA), a potent SR Ca²⁺ pump inhibitor,<br />when this agents were used at submaximal concentrations. However, when CPA was used at a maximal concentration, ryanodine had no additional effect. Ryanodine at concentrations of 10⁻⁹ to 5x10⁻⁴ M, did not significantly change the Ca²⁺ release rate. In Ca²⁺ release experiments, no functional Ca²⁺ channel was observed, but the data are consistent with an inhibition of the SR Ca²⁺ pump at high ryanodine concentrations.</p> <p>My study provided the first direct evidence ofthe existence ofryanodine receptors located on smooth muscle SR which may represent a CICR channels. Since another type of Ca²⁺ channel, IP₃-induced Ca²⁺ release channel (IICR), has also been identified in different smooth muscles, the varied levels of ryanodine binding site observed in the present study may be correlated to the ratio of occurrences of the two types of Ca²⁺ channel. This suggests that different excitation-contraction coupling mechanisms may exist in different tjpes of smooth muscle depending on different expression of these two channels. The present study also suggests that ryanodine, at higher concentrations, may inhibit the Ca²⁺ pumps located on SR. Suitable conditions for membrane isolation and Ca²⁺ transport experiment must be defmed to restore a functional Ca²⁺ release channel in smooth muscle, in order to study the functional effect of ryanodine on this channel.</p> / Doctor of Philosophy (PhD)

<p>A strategy to investigate the effect of two exercise modes upon patients with chronic airflow obstruction (CAD) is developed. The difficulties in defining and diagnosing the various pathological entities covered by the umbrella term CAO are discussed. Following a review of the published studies of endurance exercise in the before mentioned patient population a clinical problem is identified. Cycle ergometer exercise and voluntary hyperventilation were the two modalities chosen to be investigated. A 2² factorial design is selected in order that both modalities may be efficiently studied, singly and in combination, with the inclusion of a placebo exercise group.</p> <p>A statistical method is depcribed for measuring agreement between two technicians conducting a test identifying the diagnostic inclusion criteria. An additional criterion for entry into the study will be inclusion of only those patients who are particularly likely to maintain the randomly assigned maneuver. This will be determined by the response to carried out a pre-experimental sequence of three weekly test events carried out current to a CAO stabilization period. The intensity of the exercise will be established using a standardized progressive exercise test and a maximum sustainea ventilatory capacity procedure. The choice of the three dutcomes was based upon a more total definition of rehabilitation. The three primary outcomes are endurance as measured by both a twelve minute walking test and a progressive multistage treadmill test. The patients' perception of their social, emotional and physical function in response to the exercise regimen is additionally measured using a health index questionnaire.</p> / Doctor of Philosophy (PhD)

<p>Herpes simplex virus (HSV) is unusual in its ability to cause recurrent infection in a host with an ostensibly competent immune system. This thesis describes two mechanisms by which HSV evades the cellular immune response, which contribute to its ability to persist.</p> <p>Human cells infected with HSV inhibit lysis by natural killer (NK) and lymphokine-activated killer (LAK) cells. Contrary to the conclusions of previous workers, this effect is due to infection of the lytic effector cells, as shown by experiments with mutant HSV and by immunofluorescence staining of effector cells in contact with HSV-infected targets. The inhibition of lytic effector cells by HSV by cell-to-cell spread may play an important role late in the infectious cycle.</p> <p>Early in the infectious cycle, infectious virus is not present and the above mechanism cannot take effect. Instead, HSV imposes a block on the cellular pathway which presents antigen to CD8⁺ cytotoxic T lymphocytes. The result is that HSV-infected human fibroblasts show abnormalities of their class I major histocompatibility complex (MHC class I) similar to those seen in antigenpresenting mutant cell lines: the MHC class I is retained within the ER and is misfolded and unstable, implying that peptide is not associated with the heavy chain/β₂-microglobulin complex. Experiments with mutant and recombinant viruses established that this effect is due to an HSV irnmediate-early protein, ICP47, and showed that cells expressing ICP47 are not efficiently recognized and lysed by CD8+ CTL. Since ICP47 is not detectably membrane-associated, it presumably affects some cytoplasmic component of the antigen-processing pathway. One candidate for this cellular target is a small (8.5 - 9 kDa) protein, which, like ICP47, is located within the cytoplasm and nucleus of certain cell types. This protein was detected by its ability to bind to a protein consisting of ICP47 fused to the carboxy terminus of glutathione S-transferase (GST).</p> / Doctor of Philosophy (PhD)

The major focus of this project was cloning and sequencing the α-1 sub-unit of Na+/K+-ATase gene in bovine corneal endothelium. The Na+/K+-ATase, also called the Na+ pump, is a crucial transmembrane protein. By transporting water and ions from and through the cornea into the aqueous humor, it is responsible for maintenance of structural integrity, corneal hydration and thereby transparency of the cornea. The Na+ pump is characterized by a complex molecular heterogeneity that results from differential association of multiple isoforms of both a (the catalytic) and [3 (glycoprotein) sub-units. In the corneal endothelium, α-1 α-2, β-l and β-2 sub-units are expressed. Pathological and mechanical causes can disrupt the endothelial morphology and deregulate the pump function leading to corneal swelling and opacity. Recent works have focused on the study of pump expression, and the influence of different factors on the upregulation of its expression. For example, hypersaline and hyperosmotic treatment significantly increases pump expression.

Generation of large volumes to cover an existing soft tissue defect is often complicated by the lack of available tissue. The current options for soft tissue reconstruction include local and free flaps, collagen fillers, traditional fat grafting and other synthetic soft tissue fillers. But they all have limitations. Recently, a lot of interest has been generated regarding the use of human adipose derived stem cells for engineering a biocompatible soft tissue construct. Give their ready availability, viability and plasticity they appear to be the ideal building blocks for a cell based soft tissue construct. We find that these cells are easy to isolate in large numbers, easy to maintain in culture and capable of multi-lineage differentiation. hASC's are readily adherent to collagen based scaffolds and these function as the ideal cell delivery matrix. Since most wound beds are ischemic and hypoxic, changes in gene expression of hASC's was studied in conditions of hypoxia and serum deprivation. Microarray PCR results demonstrate the up regulation of 23 angiogenic genes including VEGFC, ANPEP, CXCL6, ANGPLT4 and CXCL5 in conditions of hypoxia. However, this angiogenic response was blunted with the presence of serum starvation in addition to hypoxia. Hence we chose to investigate methods to increase the primary neovascularization of a tissue engineered construct. Our hypothesis was that Europium Nano rods (belonging to the lanthanide series of heavy metals) would increase the angiogenic potential of hASC's. Results of a chick embryo chorioallantoic membrane assay demonstrate that Europium Nano rods potentiate the angiogenic effects of Vascular Endothelial Growth Factor (VEGF) when incorporated in hASC's. These rods are readily incorporated in hASC's by endocytosis and do not affect viability. Hence, we conclude that Europium Nano rods can function as a reliable, nontoxic extrinsic angiogenic stimulus. Further studies are needed to evaluate the 1) effects of ENR's on stem cell plasticity 2) effects on gene expression and 3) further investigate the fate of ENR's with repeated cell division.

The dosing of combination therapies is commonly undertaken empirically by practicing physicians, and there is a lack of a coherent algorithm to approach the problem of combination dosing. Current methods of evaluating multiple drug combinations in clinical trials generally do not provide information regarding the location of more effective dosages when the combination is not found to differ from the standard, even though the absence of a difference does not necessarily mean the new combination is ineffective. Additionally, if a new combination is found to be more effective, often a large proportion of the subjects has not benefited from the trial. This may lead to problems with patient enrollment and adherence to the study protocol, and even with early stopping rules, the time patients spend on inferior treatments may have lasting detrimental effects. This paper describes an evolutionary operation (EVOP) direct-search procedure to titrate combination doses within individual patients. The Nelder-Mead simplex direct-search method is used to titrate a combination of drugs within individual subjects. Desirability functions are incorporated to define the main response of interest and additional responses or constraints. Statistical methodology for determining whether the titrated treatment combination has resulted in an improvement in patient response and for evaluating whether a therapeutic synergism exists is developed. Inferences can be made about the efficacy of the combination or about the individual drugs that comprise the combination. This approach allows every patient the potential to benefit from the combination under study and permits the consideration of multiple endpoints simultaneously.

Parvalbumin is an abundant divalent cation binding protein of fast vertebrate skeletal muscle. Its proposed function is to sequester calcium after contraction, thus facilitating relaxation. Calcium and magnesium equilibrium dissociation constants and instantaneous rate constants of parvalbumins from two Antarctic teleosts (Gobionotothen gibberifrons and Chaenocephalus aceratus), two temperate zone teleosts (Cyprinus carpio and Micropterus salmoides), and one Arctic teleost (Boreogadus saida) were determined to assess potential differences in protein function. PV was isolated by homogenization followed by gel filtration and ion exchange chromatography. Sample purity was checked by 2-D PAGE. Dissociation constants were determined by a competitive binding assay between parvalbumin and either fluo-3 or Magnesium Green. Thermodynamic parameters were determined by calorimetry. Instantaneous rate constants were determined by stopped-flow spectrometry. Deduced amino acid sequences were also determined for several teleost parvalbumins. Functional data showed that parvalbumins from different teleost fish can exhibit markedly different patterns of thermal sensitivity. However, a general pattern of conservation for several binding parameters at native temperature was observed. Sequence data indicated that the major structural features, including the coordinating residues of the binding loops, were conserved in parvalbumins. Substitutions leading to variability of thermal function are likely to have occurred outside the binding loops of the protein. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Degree Awarded: Fall Semester, 2005. / Date of Defense: September 19, 2005. / Parvalbumin, Muscle, Temperature / Includes bibliographical references. / Timothy S. Moerland, Professor Directing Dissertation; Timothy M. Logan, Outside Committee Member; P. Bryant Chase, Committee Member; W. Ross Ellington, Committee Member; Betty J. Gaffney, Committee Member.

Arterial LDL deposition is a key step in initiating atherosclerosis. Saturated fat (SAT)-enriched diets increase arterial LDL deposition, total and selective LDL-cholesterol uptakes, and arterial lipoprotein lipase (LpL). Because consumption of n-3 fatty acids (FA), such as EPA and DHA, potentially reduces the risk for cardiovascular disease (CVD), we investigated the mechanisms whereby dietary FA, metabolic syndrome and LpL interact to influence arterial lipid uptake in atherosclerosis. We hypothesized that specific FA, SAT versus n-3, alter the recruitment of different cell populations to the arterial wall and modulate arterial LpL levels and distribution, which in turn affects the development of atherosclerosis. To test these hypotheses, we carried out a series of dietary feeding studies in different mouse models to determine the effects of saturated versus n-3 FA on LDL-cholesterol delivery and arterial LpL levels. The data presented in this thesis demonstrate that high levels of saturated FA and insulin resistance contribute to accumulation of macrophages that secrete LpL and thus favor the development of atherosclerosis. Furthermore, deficiency of LpL in the arterial wall reduced aortic macrophage populations and macrophage infiltration. The reconstitution of macrophage expressing LpL in LpL knockout models led to increases in macrophage populations in the arterial wall. Macrophage-associated LpL may increase "anchoring" of LDL and macrophages. n-3 FA decrease the presence of inflammatory macrophages and LpL; hence are associated with less arterial cholesterol delivery, inflammation and atherosclerosis. In an atherosclerosis-prone mouse model (LDLR-/-), gradual replacements of SAT with n-3 ameliorates abnormal lipid profiles and atherosclerosis. Thus, n-3 FA decrease risk for CVD in part by decreasing arterial macrophage-associated LpL. Interacting factors, such as PPARβ/δ, likely play an important role in regulation of arterial-wall LpL levels in response to FA. Increased arterial Foxo1 expression can contribute to decreased PPARα levels in the group fed n-3 FA. The levels of GPIHBP1 in the arterial wall correlate with arterial macrophage number. This indicates the existence of other pathways important in mediating changes in LpL levels and arterial lipid deposition. These studies as described here defined 'novel' mechanisms underlying the potential of n-3 FA to decrease risk for CVD.

Obesity is on the rise, and its associated comorbidities and health care costs are tremendous. A contributing factor to chronic obesity is binge eating disorder (BED), which is prevalent in 20 to 30 percent of the morbidly obese population, but the distinction between obesity versus obesity with BED is still unclear. The present dissertation project investigated forty two adult men and women, thirteen obese + BED and twenty nine obese controls for multiple psycho-behavioral constructs (rigid dietary restraint, disinhibition, anxiety, and behavioral activation/behavioral inhibition). On a different day, following a 12-hour fast, the participants consumed a fixed liquid meal, and their brain function examined while images of high energy food (e.g. pizza and cakes), low energy food (e.g. cucumber and tomato) and control items (i.e. office supplies) presented to them on a screen. Using a whole brain analysis approach, functional brain activity in response to: 1/food versus nonfood, and 2/high energy food versus low energy food revealed eight brain areas significantly different between the groups: for 'food versus nonfood', activated were seven areas functionally involved in the integration of somatosensory experience with internal state, processing of sensations, cognitions, thoughts, and emotions, integration of sensory functions and memory, visual object recognition and motion, visual - somatosensory functions and associations, integration of emotional value with a sensory stimulus, mediation of motivation and expectancy for outcomes, and the integration of diverse sensory information and visuo-spatial cognition. . One area significantly differed between the groups in response to the comparison of 'high energy food versus low energy food'. This area is functionally involved in thought, cognition, movement, planning, and motor behaviors in response to emotions and drives Thus, in response to cues representing binge-triggers, obese + BED showed greater visual attention, emotional, motivational and reward processing, as well as motor planning of future actions and heightened somatosensory experience, compared with the obese group. Scores on the 'disinhibition' scale were significantly higher in the obese + BED group compared with the obese. Correlation between 'disinhibition' scores and brain activation results in each group showed significant differences between the groups in two brain areas: right anterior cingulate gyrus-Brodmann area #32, and the left postcentral gyrus. Scores on the Behavioral Activation Scale (reward drive) were significantly lower in the obese + BED group, but the correlations between brain activation and scores on this scale did not differ between the groups. To sum the results altogether, the obese + BED may be marked by hyperactive visual-attentional-emotional- and cognitive processing of cues representing binge-triggers, with heightened somatosensory response. The psycho-behavioral construct of 'disinhibition' highly characterizes BED, and its neurobiological substrates may include the right anterior cingulate cortex-Brodmann area #32 and left postcentral gyrus. Reduced reward responsiveness in obese + BED may reflect weak 'liking' response to food, but this behavioral construct and its' relationship to BED are still inconclusive. Future studies may use the results of this dissertation project to further investigate frequent binge eating in the absence of compensatory behaviors in the obese population.